Tumor necrosis factor (TNF)alpha plays a key role in regulation of intestinal growth, development and the pathogenesis of inflammatory bowel diseases by several critical signal transduction pathways, including the extracellular signal regulated kinase (ERK1/ERK2)/mitogen activated protein (MAP) kinase. Preliminary studies have shown expression of the ceramide-regulated dominant-negative, kinase inactive (ki) kinase suppressor of Ras (KSR) inhibits TNFalpha-induced ERK1/ERK2 activation. Furthermore, kiKSR expression converts TNFalpha from anti-proliferative to apoptotic in intestinal epithelial cells, implicating KSR as a key mediator of TNFalpha signaling. This proposal is designed to test the hypothesis that KSR is an essential regulatory molecule in TNFalpha stimulated ERK/MAP kinase activation. Three specific aims are proposed for study. Aim 1. What is the ceramide regulatory domain on KSR? Aim 2. What regulatory domains of KSR are required for ERK1/ERK2 activation by TNFalpha in intestinal epithelial cells? Aim 3. What proteins associate with the KSR regulatory domains in ERK1/ERK2 activation by TNFalpha? A variety of methods will be employed to achieve these Specific Aims, including site-directed and deletion mutagenesis, protein kinase assay, yeast two-hybrid assay and expression of GST-fusion proteins. The findings from these studies will have broad implications on intestinal biology, from normal growth and development to the pathogenesis of inflammatory bowel disease.